Influenza A virus vaccines and inhibitors

ABSTRACT

The present invention includes compositions and methods related to the structure and function of the cellular polyadenylation and specificity factor 30 (CPSF30) binding site on the surface of the influenza A non-structural protein 1 (NS1). Specifically, critical biochemical reagents, conditions for crystallization and NMR analysis, assays, and general processes are described for (i) discovering, designing, and optimizing small molecule inhibitors of influenza A (avian flu) viruses and (ii) creating attenuated influenza virus strains suitable for avian and human flu vaccine development.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 60/741,764, filed Dec. 2, 2005 and Ser. No. 60/852,361 filed Oct.16, 2006, the entire contents of which are incorporated herein byreference. This application is related to U.S. patent application Ser.No. 11/566,216, filed on Dec. 2, 2006.

STATEMENT OF FEDERALLY FUNDED RESEARCH

This invention was made with U.S. Government support under Contract Nos.AI-11772 awarded by the NIH. The government has certain rights in thisinvention.

TECHNICAL FIELD OF THE INVENTION

The present invention relates in general to the field of anti-viralassays and molecules, and more particularly, to compositions and methodsfor developing, isolating and characterizing novel Influenza A virusinhibitors and vaccines.

INCORPORATION-BY-REFERENCE OF MATERIALS FILED ON COMPACT DISC

The present application includes a TABLE filed electronically viaEFS-Web that includes a file named NS1A_F2F3. The table was lastmodified Dec. 1, 2006, 2006 at 4:49 PM and includes 252,952 bytes.

BACKGROUND OF THE INVENTION

Without limiting the scope of the invention, its background is describedin connection with Influenza virus.

Influenza A and B viruses cause a highly contagious respiratory diseasein humans resulting in approximately 36,000 deaths in the United Statesannually (Wright and Webster, 2001; Prevention, 2005). These annualepidemics also have a large economic impact, and cause more than 100,000hospitalizations per year in the United States alone. Influenza Aviruses, which infect a wide number of avian and mammalian species, areresponsible for the periodic widespread epidemics, or pandemics, thathave caused high mortality rates (Wright and Webster, 2001). The mostdevastating pandemic occurred in 1918, which caused an estimated 20 to40 million deaths worldwide (Reid et al., 2001). Less devastatingpandemics occurred in 1957 and 1968. Influenza B virus infectionscomprise about 20% of the yearly cases, but influenza B virus, whichappears to infect only humans, does not cause pandemics (Wright andWebster, 2001).

Influenza A and B viruses contain negative-stranded RNA genomes, whichare in the form of eight RNA segments (Lamb and Krug, 2001). Most, butnot all, of the corresponding genome RNA segments of influenza A and Bviruses encode proteins of similar functions. Here we will focus oninfluenza A virus. The three largest genome segments encode the threesubunits of the polymerase, PB 1, PB2 and PA. The segment encoding PB 1also encodes a small nonstructural protein, PB1-F2, which has apoptoticfunctions. The middle-sized segments encode the hemagglutinin (HA), thenucleocapsid protein (NP) and the neuraminidase (NA). HA, the majorsurface protein of the virus, binds to sialic acid-containing receptorson host cells, and is the protein against which neutralizing antibodiesare produced. NP protein molecules are bound at regular intervals alongthe entire length of each of the genomic RNAs to form ribonucleoproteins(RNPs), and also have essential functions in viral RNA replication. TheNA viral surface protein removes sialic acid from glycoproteins. One ofits major functions is to remove sialic acid during virus budding fromthe cell surface and from the HA and NA of the newly assembled virions,thereby obviating aggregation of the budding virions on the cellsurface. The seventh genomic RNA segment encodes two proteins, M1(matrix protein) and M2. The M1 protein underlies the viral lipidmembrane, and is thought to interact with the genomic RNPs and with theinner (cytoplasmic) tails of the surface proteins, e.g., HA and NA. TheM2 protein is an ion channel protein that is essential for the uncoatingof the virus. The smallest segment encodes two proteins, NS1A and NS2.The NS2 protein mediates the export of newly synthesized viral RNPs fromthe nucleus to the cytoplasm. The NS1A protein is a multi-functionalprotein not that is not incorporated into virion particles (hence thedesignation “non structural”), and is discussed below.

The primary means for controlling influenza virus epidemics has beenvaccination directed primarily against HA (Wright and Webster, 2001).However, the antigenic structure of the HA of influenza A virus canundergo two types of change (Wright and Webster, 2001). Antigenic driftresults from the selection of mutant viruses that evade antibodiesdirected against the major antigenic type of the HA circulating in thehuman population. Mutant viruses are readily generated because the viralRNA polymerase has no proof-reading function. Because of antigenicdrift, the vaccine has to be reformulated each year. Antigenic shift inHA results from reassortment of genomic RNA segments between human andavian influenza A virus strains, resulting in a new (potentiallypandemic) virus encoding a novel avian-type HA that is immunologicallydistinct. The human population has little or no immunological protectionagainst such a new virus. The viruses containing the H2 and H3 HAsubtypes that caused pandemics in 1957 and 1968, respectively, resultedfrom the reassortment of avian and human genomic RNA segments (Wrightand Webster, 2001). The HA of influenza B viruses undergoes antigenicdrift, but not antigenic shift, because influenza B viruses do not havenon-human hosts.

Pandemic influenza A viruses can also apparently arise by a differentmechanism. It has been postulated that the 1918 H1 pandemic strainderived all eight genomic RNAs from an avian virus, and that this virusthen underwent multiple mutations in the process of adapting tomammalian cells (Reid et al., 2004; Taubenberger et al., 2005). H5N1viruses, which have already spread from Asia to Europe and Africa,appear to be undergoing this route for acquiring pandemic capability(Horimoto and Kawaoka, 2005; Noah and Krug, 2005). These viruses, whichhave been directly transmitted from chickens to humans, contain onlyavian genes, and are highly pathogenic in humans. The human mortalityrate has been high, approximately 55% (WHO, 2006). H5N1 viruses have notyet acquired the ability for efficient transmission from humans tohumans. Recent studies indicate that efficient human transmission willrequire more than the acquisition of the ability of HA to bind to humansialic acid receptors in the upper respiratory tract of mammalianorganisms (Maines et al., 2006). However, at least one H5N1 gene, thePB2 gene, has already undergone adaptation to mammalian cells (Hatta etal., 2001). The vast majority of pathogenic H5N1 viruses have acquired alysine at position 627 in the PB2 protein, in place of the glutamic acidthat is found at this position in avian viruses. The presence of lysineat this position apparently enhances virus replication in mammaliancells, but the mechanism of enhancement has not been established(Crescenzo-Chaigne et al., 2002; Shinya et al., 2004).

Effective control of a H5N1 pandemic will require the use of antiviraldrugs because it is not likely that sufficient amounts of an effectivevaccine will be available, particularly in the early phase of afast-spreading pandemic (Ferguson et al., 2005; Longini et al., 2005;Ferguson et al., 2006; Germann et al., 2006). Antivirals can bestockpiled, and if appropriately used, should limit the spread ofpandemic influenza virus. The strategies that have been proposed for theuse of antivirals to stem a H5N1 pandemic would also be expected to leadto more effective use of antivirals during annual influenza epidemics.Currently, there are two classes of antiviral drugs. One class,amantadine/rimantidine, is directed against the M2 ion channel ofinfluenza A viruses (Pinto et al., 1992; Wang et al., 1993; Chizhmakovet al., 1996). Virus mutants resistant to this class of drugs rapidlyemerge (Cox and Subbarao, 1999; Suzuki et al., 2003), and many of thehuman isolates of H5N1 viruses are already resistant to these drugs(Puthavathana et al., 2005). The other class of drugs is directed at NA,and is effective against both influenza A and B viruses (von Itzstein etal., 1993; Woods et al., 1993; Ryan et al., 1994; Gubareva et al., 1995;Kim et al., 1997; Mendel et al., 1998). However, H5N1 viruses that arepartially, or completely, resistant to the NA inhibitor oseltamivir havebeen reported (de Jong et al., 2005; Le et al., 2005). The emergence ofH5N1 viruses to these two classes of antiviral drugs highlights the needfor additional antiviral drugs against influenza virus.

SUMMARY OF THE INVENTION

The present invention includes the structure and function of thecellular polyadenylation and specificity factor 30 (CPSF30) bindingsite, or “binding epitope”, on the surface of the influenza Anon-structural protein 1 (NS1). When referring to influenza A, theprotein is referred to herein as NS1A. The NS1 protein of influenza Avirus will be designated as the NS1A protein to distinguish it from theNS1 protein encoded by influenza B virus, which will be designated asthe NS1B protein. Several fragments of the NS1A protein were identified,from various influenza strains, that provide high-level expression andsolubility in E. coli expression systems. The present invention alsoincludes expression systems and protocols for producing large quantitiesof the previously identified F2F3 fragment of human CPSF30, which bindsto the NS1A effector domain (Twu et al., 2006). These fragments of theNS1A protein and of CPSF30 provide high quality NMR spectra, suitablefor lead compound optimization. Combining these reagents andinformation, we have designed a gel filtration assay suitable forcharacterizing complex formation between NS1A and CPSF30 (or fragmentsthereof), as well as a fluorescence polarization (FP) assay ofNS1A-CPSF30 interactions that is suitable for high throughput screeningfor lead compounds to develop antiviral drugs. Using these reagents,complexes of the tetrameric complex between the NS1A effector domain andF2F3 were also generated. We have also developed a process forcrystallizing this complex. These crystals were used to determine the 3Dstructure of the NS1A effector domain:F2F3 complex, and to definitivelyidentify for the first time all the NS1A amino acids that comprise aportion of the CPSF30 binding epitope in the NS1A protein. Thesereagents and structural data, together with specific site-directedmutagenesis data, have allowed us to define a structure-based processfor high-throughput screening of inhibitors and lead optimization thatwill allow development of influenza A antiviral drugs. Also disclosed isa process using these reagents and structural data to develop attenuatedstrains of influenza A suitable for use as animal (e.g. avian) or humanvaccines.

Using the present invention it is possible for antiviral drug(s) to bedirected at a viral target that differs from that of currently availableinfluenza antivirals. Novel approaches are important because influenza Aviruses, including avian influenza virus, are developing resistance toexisting antiviral drugs. The present invention also describes a processfor developing vaccines using structural information that has not beenpreviously disclosed. A vaccine using live attenuated strains ofinfluenza A virus is expected to provide better protection than avaccine using inactivated virus. The present invention may also be usedto develop, isolate and characterize antiviral drugs and live attenuatedvirus vaccine for both seasonal and pandemic influenza virus infections.

More particularly, the present invention includes a complex of the F2F3fragment of CPSF30 with the portion of the NS1A protein of influenza Avirus comprising amino acid residues 85 to 215 (NS1A (85-215)). Thecomplex can be used in a method of identifying an inhibitor of influenzaA virus by preparing a reaction system comprising at least a portion ofNS1A of an influenza A virus, the F2F3 fragment of CPSF30, and acandidate compound; and detecting binding between the at least a portionof NS1A and F2F3, wherein reduced binding in the presence of thecandidate drug relative to the control is indicative of activity of thecompound against influenza virus. Examples of the NS1A fragments arelisted in Table 2, and may also include the full length NS1A, andmutations thereof. The skilled artisan will recognize that the NS1Aprotein fragment may be prepared from other strains of influenza Aviruses, e.g., human influenza A virus, a bovine influenza A virus, anequine influenza A virus, a porcine influenza A virus, an avianinfluenza A virus, an avian H5N1 viral strain.

For use with the present invention, the CPSF30 may be, e.g., a fulllength CPSF30, F3 zinc-finger doman, an F2F3-F2F3 tandem duplex, anF1F2F3 fragment, a soluble fragment of CPSF30, combinations and mutantsthereof. Detection of the binding may be by, e.g., a high throughputscreening assay, X-ray crystallography, nuclear magnetic resonance,analytical gel filtration, and combinations thereof. Other methods ofdetecting the interaction of the NS1A and CPSF30 (or portions thereof)may be, e.g., fluorescence resonance energy transfer, fluorescencepolarization, immunofluorescence, chemiluminescence, radioimmunoassays,enzyme linked immunosorbent assays, mass spectrometry, and combinationsthereof. For example, fluorescence polarization may use either afluorescent labeled CPSF30, variant or fragment thereof or a fluorescentlabeled NS1A protein, variant or fragments thereof.

The present invention also includes a process for using 3D(three-dimensional) information of the CPSF30 binding epitope on theinfluenza A virus NS1A protein for identifying inhibitors of aninfluenza A virus by obtaining concurrently the coordinates for themolecular position of at least a portion of an influenza NS1A proteinand at least a portion of a CPSF30 protein and designing a molecule thatwill fit between the influenza NS1A protein and the CPSF30 protein basedon the coordinates. The portion of the NS1A protein and the portion ofthe CPSF30 protein will generally be selected to provide high qualityNMR spectra, e.g., the NS1A (85-215) fragment and the F2F3 fragment maybe selected to form a tetrameric complex.

In another example, the NS1A (85-215) fragment and the F2F3 fragment mayform a tetrameric complex that is crystallized. Other examples may usean NS1A (85-215) fragment and the F2F3 fragment form a tetramericcomplex that is crystallized and is used to determine the 3D structureof the NS1A (85-215):F2F3 complex using X-ray crystallography. Themethod may also include the step of identifying the NS1A amino acidsthat constitute the CPSF30 binding portion of the NS1A protein.

Yet another embodiment is a method of designing an inhibitor compound ofan influenza A virus based on computational methods, wherein theinhibitor that binds at least one residue within the set of residuescorresponds to the structure, using the crystal structure and itscoordinates. The design method may be facilitated by using NMR spectraof samples of NS1A protein fragments, NMR spectra of samples of NS1A(85-215) or equivalent constructs thereof. The method may furtherinclude the step of optimizing the design of the molecule that fitsbetween the NS1A (85-215) protein and the CPSF30 protein using X-raycrystallography, and synthetic chemistry. The method of designing aninhibitor compound may also include step of optimizing the design of themolecule that fits between the NS1A (85-215) protein and the CPSF30protein using X-ray crystallography, NMR, synthetic chemistry andcombinations thereof. Another embodiment of method of designing aninhibitor compound may also include, optionally or in combination one ormore of the following: modifying the F2F3 structure of CPSF30, or anysubstructure, based on its conformation bound to NS1A to rationallydesign one or more small molecule inhibitors; modifying the F2F3structure of CPSF30, or any substructure, to rationally design one ormore small molecule inhibitors based on its conformation bound to NS1Aof the F2F3 aromatic rings, the sidechain of residue Lys101, orcombinations thereof; modifying the F2F3 structure of CPSF30, or anysubstructure, to rationally design one or more small molecule inhibitorsby virtual screening; using the three-dimensional structure of the NS1Abinding pocket to select one or more small molecule inhibitors or usingthe three-dimensional structure of the NS1A binding pocket to select oneor more small molecule inhibitors by virtual screening. Any fragmentsof, full length, and strains of NS1A, and fragments of, full length, andstrains of CPSF30 and combinations thereof may be used in conjunctionwith the method of designing an inhibitor compound.

Another embodiment of the present invention includes a process forengineering a live attenuated influenza A virus vaccine by mutation ofspecific residues in the CPSF30-binding epitope of the NS1A proteinbased on the 3D structure of the complex formed between NS1A (85-215)and F2F3, a tetramer interface and combinations thereof. Specificexamples of residues at the tetramer interface site include, e.g., F103,L105, M106 and equivalents thereof. Specific examples of residues at theCPSF30-binding epitope include, e.g., M106, K110, I117, I119, Q121,D125, V180, G183, G184, W187 and equivalents thereof. The process mayfurther include mutating the double-strand RNA binding epitope.

Another embodiment of the present invention includes an attenuatedinfluenza A virus vaccine that includes one or more of eight viral RNAsegments, wherein a viral RNA segment eight has a first mutation thatcauses a substitution of a first amino acid corresponding to an aminoacid of SEQ. ID. NO. 1 at a position F103, L105, M106, K110, I117, I119,Q121, D125, L144, V180, G183, G184, or W187; wherein the first mutationdecreases the CPSF30 binding ability of the NS1A protein. In oneembodiment, at least a second mutation anywhere in the NS1A protein. Thevaccine may also include at least second mutation causes a substitutionof at least a second amino acid corresponding to an amino acid of SEQ IDNO.: 1 at a position F103, L105, M106, K110, I117, I119, Q121, D125,L144, V180, G183, G184, W187 and equivalents thereof. Alternatively, thevaccine may also include at least second mutation with a substitution ofat least a second amino acid corresponding to an amino acid of SEQ IDNO.: 1 at a position T5, P31, D34, R35, R38, K41, G45, R46, and T49 at adsRNA binding epitope and equivalents thereof. Alternatively, thevaccine may also include at least second mutation with at least secondmutation causes a substitution of at least a second and third amino acidcorresponding to an amino acid of SEQ. ID. NO. 1 at a position T5, P31,D34, R35, R38, K41, G45, R46, and T49 at a dsRNA binding epitope and/orF103, L105, M106, K110, I117, I119, Q121, D125, L144, V180, G183, G184,W187 and equivalents thereof. The influenza virus is a may be acold-adapted influenza virus and will generally be selected to elicit animmune response. Non-limiting examples of influenza virus that may beused to make the attenuated vaccine may be is selected from, e.g., ahuman influenza A virus, a bovine influenza A virus, an equine influenzaA virus, a porcine influenza A virus, an avian influenza A virus, anavian H5N1 viral strain.

The present invention also includes a pharmaceutical composition havinga live attenuated influenza A virus vaccine by mutation of specificresidues in the CPSF30-binding epitope of the NS1A protein based on the3D structure of the complex formed between NS1A (85-215) and F2F3, atetramer interaction site and combinations thereof and apharmaceutically acceptable carrier or diluent. The vaccine may be usedin a method of prophylaxis of a disease condition caused by theinfluenza virus by administering to a subject in need thereof atherapeutically effective amount of a live attenuated influenza A virusvaccine by mutation of specific residues in the CPSF30-binding epitopeof the NS1A protein based on the 3D structure of the complex formedbetween NS1A (85-215) and F2F3, a tetramer interaction site, the dsRNAbinding epitope and combinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of thepresent invention, reference is now made to the detailed description ofthe invention along with the accompanying figures and in which:

FIGS. 1A and 1B are graphs that show the effects on the growth of arecombinant influenza A virus resulting from mutations in its ofmutations on NS1A protein that relate to the ability of the NS1A to bindto CPSF30;

FIG. 2 shows NMR spectra of certain NS1A effector domains, demonstratingthat such data can be used for structure-function analysis, inhibitordiscovery and optimization, and in validating the effects of mutationson the structure of the effector domain;

FIG. 3 shows the results from analytical gel filtrations assays of NS1A(85-215):F3F3 binding;

FIG. 4A shows the X-ray crystal structure of a tetrameric NS1A effectordomain and F2F3;

FIG. 4B shows the X-ray crystal structure for the Met106 residue in thecore of the tetrameric NS1A (85-215):F2F3 complex;

FIG. 5 shows the X-ray crystal structure for the F2F3 binding epitope onthe surface of the NS1A effector domain;

FIG. 6 is a comparison of 3D structures for Udorn and PR8 NS1A effectordomains; and

FIG. 7 shows the 3D structure and dimeric beta-sheet interface of thePR8 effector domain, which does not bind F2F3.

DETAILED DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

To facilitate the understanding of this invention, a number of terms aredefined below. Terms defined herein have meanings as commonly understoodby a person of ordinary skill in the areas relevant to the presentinvention. Terms such as “a”, “an” and “the” are not intended to referto only a singular entity, but include the general class of which aspecific example may be used for illustration. The terminology herein isused to describe specific embodiments of the invention, but their usagedoes not delimit the invention, except as outlined in the claims.

One of the best clues to a protein's function is its structure. Thepresent invention takes advantage of structure-based bioinformaticsplatforms in “functional genomics.” The structure-function data obtainedfrom various structural determination may be used the isolation of novelbiopharmaceuticals and/or drug targets from gene sequence informationwith greater efficiency. One way to identify the biochemical and medicalfunction of a gene product is to determine its three-dimensionalstructure. Although there are numerous examples in which the primary(i.e., linear) structure of a protein has provided key clues to itsbiochemical function, three dimensional (3D) structure determination isconsidered to be more definitive at establishing biochemical functionand mechanisms underlying these functions.

Protein structure. It is a generally accepted principle of biology thata protein's primary sequence is the main determinant of its tertiarystructure. Anfinsen, Science 181:223-230 (1973); Anfinsen and Scheraga,Adv. Prot. Chem. 29:205-300 (1975); and Baldwin, Ann. Rev. Biochem.44:453-475 (1975). For over a decade, researchers have been studying thetheoretical and practical aspects of the folding of recombinantproteins.

Generally, proteins are composed of one or more autonomously-foldingunits known as domains. Kim, et al., Ann. Rev Biochem. 59:631-660(1990); Nilsson, et al., Ann. Rev. Microbiol. 45:607-635 (1991).Multi-domain proteins in higher organisms are encoded by genescontaining multiple exons and combinatorial shuffling of exons duringevolution has produced novel proteins with different domain arrangementshaving different associated functions. Multi-domain protein may increasethe ability of higher organisms to respond to environmental challengesbecause, via recombinational events, because the genomes may readilyadd, subtract, or rearrange discrete functionalities within a givenprotein. Patthy, Cell 41:657-663 (1985); Patthy, Curr. Opin. Struct.Bio. 4:383-392 (1994); and Long, et al., Science 92:12495-12499 (1995).

Interpretation of a protein structure. Several methods have been used toelucidate the 3D structure of a given protein molecule, e.g., X-raycrystallography and Nuclear Magnetic Resonance (NMR).

X-ray crystallography. X-ray crystallography is a technique thatdirectly images molecules. A crystal of the molecule to be visualized isexposed to a collimated beam of monochromatic X-rays and the consequentdiffraction pattern is recorded on a photographic film or by a radiationcounter. The intensities of the diffraction maxima are then used toconstruct mathematically the three-dimensional image of the crystalstructure. X-rays interact almost exclusively with the electrons in thematter and not the nuclei. The spacing of atoms in a crystal lattice canbe determined by measuring the angle and intensities at which a beam ofX-rays of a given wave length is diffracted by the electron shellssurrounding the atoms. Operationally, there are several steps in X-raystructural analysis. The amount of information obtained depends on thedegree of structural order in the sample. Blundell et al. provide anadvanced treatment of the principles of protein X-ray crystallography.Blundell, et al., Protein Crystallography, Academic Press (1976), hereinincorporated by reference. Likewise, Wyckoff et al. provide a series ofarticles on the theory and practice of X-ray crystallography. Wyckoff,et al. (Eds.), Methods Enzymol. 114: 330-386 (1985), relevant portionsherein incorporated by reference. Important techniques for X-raycrystallography include methods for determining diffraction data phasesby a multiple anomalous dispersion (MAD) method (described, for example,in Hendrickson, 1991), particularly using biosynthethic enrichment withseleno-methione (SeMet), and a molecular replacement method (described,for example, in Rossmann, M. G. The molecular replacement method. ActaCryst. A46, 73-82 (1990).

Nuclear Magnetic Resonance (NMR). A general approach for the analysis ofNMR resonance assignments was first outlined by Wuthrich, Wagner andco-workers. Wuthrich, “NMR of proteins and nucleic acids” Wiley, NewYork, N.Y. (1986); Wuthrich, Science 243:45-50 (1989); Billeter, et al.,J. Mol. Biol. 155:321-346 (1982), relevant portions of each incorporatedherein by reference. For a general review of protein determination insolution by nuclear magnetic resonance spectroscopy, see Wuthrich,Science 243:45-50 (1989); Billeter et al., J. Mol. Biol. 155:321-346(1982). More recent improvements using multidimensional and tripleresonance NMR methods (described, for example, in J. Cavanagh, W.Fairbrother, A. Palmer, and N. Skelton, Protein NMR Specroscopy,Principles and Practice, 2ed Ed, Academic Press, NY, 2006, incorporatedherein), familiar to someone trained in the art, are elaborations ofthese basic principles.

As used herein, the terms “CPSF30 binding epitope” or “CPSF bindingsite” refer to that portion of the NS1 protein of influenza thatinteracts with one or more zinc fingers of the CPSF30 protein.

As used herein, the terms “NS1A-binding epitope” or “NS1A binding site”refer to that portion of the CPSF30 protein that interacts with the NS1protein of influenza A.

As used herein, the term “tetramer interface” refers to those portionsof the NS1A protein and the CSPF30 protein that interact NS1A:CPSF30complex, which can be a dimer or tetramer.

As used herein, the terms “double-strand RNA binding site” or“doubled-stranded RNA binding epitope” refer to that portion of the NS1Aprotein that interacts with double-stranded RNA, e.g., for suppressinghost innate immune response. Greater structural details regarding thedouble-strand RNA binding site is taught by the present co-inventors inPCT/US2003/036,292, “Process for Designing Inhibitors of Influenza VirusNon-Structural Protein 1,” relevant portions incorporated herein byreference and U.S. Provisional Patent Application Ser. No. 60/737,742,“Novel Compositions and Vaccines Against Influenza and Influenza BInfections,” and PTC patent application Filed Nov. 17, 2006 “NovelCompositions and Vaccines Against Influenza and Influenza B Infections,”relevant portions and tables incorporated herein by reference.

As used herein, the term “epitope” refers to a portion of a protein thatis capable of interacting with the same or another protein and that canbe define by atoms in amino acids in a linear sequence, by atoms inamino acids that are located throughout the protein (or subunitsthereof) and that come together in three dimensions to form aninteractive structure. The skilled artisan will recognize that anepitope may be a structure such as a “zinc finger,” a portion of amolecular surface between two or more polypeptides that are capable ofinteracting, and/or a “binding pocket” or “enzymatic pocket” that leadto a function or interaction of the protein.

As used herein, the following nomenclature is used to define the zincfinger domains of the CPSF30 protein that interact NS1A, namely, “F1”,“F2” and “F3” when referring to individual zinc fingers, “F1F2”, “F2F3”when referring to a pair of zinc fingers and “F1F2F3” when referring tothe three zinc fingers.

As used herein, the term “rationally design” refers to a series of stepsfor designing, e.g., initial small molecule inhibitors that may befurther refined through medicinal chemistry and/or the selection ofinhibitors that can be further modified chemically and that fit themolecular characteristics of a molecule that will meet the designcriteria for modifying the interactions between or the activity of NS1Aand CPSF30 based fully and/or at least in part on the three dimensional(3D) coordinates described herein.

As used herein, the term “virtual screening” refers to in silico design,using computer software to screen for possible binding ligands from alibrary of virtual molecules, allowing the characterization andrefinement of small molecule inhibitors that will meet the designcriteria for modifying the interactions between or the activity of NS1Aand CPSF30 based fully and/or at least in part on the three dimensional(3D) coordinates described herein.

As used herein, the term “corresponding positions” refers to amino acidsat that are generally in the same spatial location in thethree-dimensional structures of homologous protein structures. In oneexample, a corresponding position is determined by 3D structurealignment using standard techniques known to a person skilled in theart, such as the PRISM program, described by Yang and Hong Yang, A.-S.and Honig, B. (2000) An Integrated Approach to the Analysis of Sequenceand Structure. I. Protein Structural Alignment and a QuantitativeMeasure for Protein Structural Distance. J. Mol. Biol. 301: 665-678;Yang, A.-S. and Honig, B. (2000) An Integrated Approach to the Analysisof Sequence and Structure. III. A Comparative Study of SequenceConservation in Protein Structural Families Using Multiple StructuralAlignments, or the VISTAL program, described by Kolodny, R. and Honig,B. (2006) VISTAL—A New 2D Visualization Tool of Protein 3D StructuralAlignments. Bioinformatics 22:2166-2167 (e.g., using the defaultsettings for either), relevant portions incorporated herein byreference. The alignment may include the introduction of gaps in thesequences to be aligned. In the absence of 3D structures for proteinpairs, corresponding positions may be identified (until such time thatthe requisite 3D structures are available) by sequence-based alignmentusing stardard techniques known to a person skilled in the art, such asa Needleman-Wunsch dynamic programming algorithm (described inNeedleman, S. B. and Wunsch, C. D., J Mol. Biol. 48: 443-453 (1970), aGapped BLAST/PSI-BLAST algorithm (described in Altschul, et al Nucl.Acid. Res. 25: 3389-3402 (1997), or a CLUSTAL W/X multiple sequencealignment algoithm (described in Thompson, J. D., Higgins, D. G. andGibson, T. J. (1994) CLUSTAL W: improving the sensitivity of progressivemultiple sequence alignment through sequence weighting, positionspecific gap penalties and weight matrix choice. Nucl. Acid. Res.22:4673-80 (1994).) (e.g., using the default settings for eitherrelevant portions incorporated herein by reference. Correspondingposition alignment(s) may also include the introduction of gaps in thesequences to be aligned.

Further examples of “corresponding positions are taught by the presentco-inventors in PCT/US2003/036,292, “Process for Designing Inhibitors ofInfluenza Virus Non-Structural Protein 1,” relevant portionsincorporated herein by reference and U.S. Provisional Patent ApplicationSer. No. 60/737,742, “Novel Compositions and Vaccines Against Influenzaand Influenza B Infections”, and PTC patent application Filed Nov. 17,2006 “Novel Compositions and Vaccines Against Influenza and Influenza BInfections,”, relevant portions and tables incorporated herein byreference.

Non-limiting examples of numerous influenza A strains may be used withthe present invention including those strains that are readily availableor will become available as they emerge in human populations. Examplesof influenza A strains for use and analysis with the present inventionmay include A/Memphis/8/88, A/Chile/1/83, A/Kiev/59/79, A/Udorn/307/72,A/NT/60/68, A/Korea/426/68, A/Great Lakes/0389/65, A/Ann Arbor/6/60,A/Leningrad/13/57, A/Singapore/1/57, A/PR/8/34, A/Vietnam/1203/04,A/HK/483/97, A/South Carolina/1/181918 (the 1918 pandemic virus H1N1strain), and A/WSN/33. The sequences of these strains are available fromGenBank, CDC and viral stock may be available from the American TypeCulture Collection, Rockville, Md. or are otherwise publicly available.

As used herein, the term “variant” gene or protein refers to a gene orprotein that differs from the wild type gene or protein by way ofnucleotide or amino acid substitution(s), addition(s), deletion(s), andcombinations thereof. Depending on the context, the term mutant is alsoused to describe variations from the wild-type sequence that arenatural, introduced at random and/or introduced into a gene or proteinsequence specifically. Likewise, a fragment is used to describe afragment of the complete length of the translated polypeptide, whethernatural or not. Often, the skilled artisan will design fragments thatinclude or avoid certain amino acid that may affect, e.g., synthesis orcause steric hindrance or other design requirements. When designingalternate peptide constructs with enhanced anti-viral properties,substitutions may be used which modulate one or more properties of themolecule. Variants typically include the exchange of one amino acid foranother at one or more sites within the peptide. For example, certainamino acids may be substituted for other amino acids in a peptidestructure in order to enhance the interactive binding capacity of thestructures. Certain amino acid substitutions can be made in a proteinsequence (or its underlying DNA coding sequence) to create a peptidewith superior functional characteristics. In particular, those changesthat enhance the amphipathic, α-helical nature may be desired.

As used herein, the term “candidate compound” refers to any moleculethat may inhibit influenza viral growth. The candidate substance may bea protein or fragment thereof, a small molecule, or even a nucleic acidmolecule. Various commercial sources of small molecule libraries meetthe basic criteria for useful drugs in an effort to “brute force” theidentification of useful candidate compounds. Screening of suchlibraries, including libraries generated combinatorially (e.g., peptidelibraries, aptamer libraries, small molecule libraries), is a rapid andefficient way to screen large number of related (and unrelated)compounds for activity. Combinatorial approaches also lend themselves torapid evolution of potential drugs by the creation of second, third andfourth generation compounds modeled of active, but otherwise undesirablecompounds. Candidate compounds may be screened from large libraries ofsynthetic or natural compounds. One example of a candidate compoundlibrary is an FDA-approved library of compounds that can be used byhumans. Synthetic compound libraries are commercially available from anumber of companies including Maybridge Chemical Co. (Trevillet,Cornwall, UK), Comgenex (Princeton, N.J.), Brandon Associates(Merrimack, N.H.), and Microsource (New Milford, Conn.) and a rarechemical library is available from Aldrich (Milwaukee, Wis.).Combinatorial libraries are available or can be prepared. Alternatively,libraries of natural candidate compounds in the form of bacterial,fungal, plant and animal extracts are also available from, for example,Pan Laboratories (Bothell, Wash.) or MycoSearch (N.C.), or can bereadily prepared by methods well known in the art. Candidate compoundsisolated from natural sources, such as animals, bacteria, fungi, plantsources, including leaves and bark, and marine samples may be assayed ascandidates for the presence of potentially useful pharmaceutical agents.It will be understood that the pharmaceutical agents to be screenedcould also be derived or synthesized from chemical compositions orman-made compounds.

The amino acid sequence of the NS1A protein of Influenza A virus,A/Udorn/72:

(SEQ ID NO.: 1)   1MDPNTVSSFQ VDCFLWHVRK RVADQELGDA PFLDRLRRDQ KSLRGRGSTL GLDIETATRA  61GKQIVERILK EESDEALKMT MASVPASRYL TDMTLEEMSR EWSMLIPKQK VAGPLCIRMD 121QAIMDKNIIL KANFSVIFDR LETLILLRAF TEEGAIVGEI SPLPSLPGHT AEDVKNAVGV 181LIGGLEWNDN TVRVSETLQR FAWRSSNENG RPPLTPKQKR EMAGTIRSEV.

The anti-viral agents disclosed herein may be used in conjunction withmethods to reduce virus growth, infectivity, burden, shed, developmentof anti-viral resistance, and to enhance the efficacy of traditionalanti-viral therapies.

The amino acid sequence of the relevant fragments of human CPSF30(UniProt id 095639):

(SEQ ID NO.: 13)   1MQEIIASVDH IKFDLEIAVE QQLGAQPLPF PGMDKSGAAV CEFFLKAACG 50  51KGGMCPFRHI SGEKTVVCKH WLRGLCKKGD QCEFLHEYDM TKMPECYFYS 100 101KFGECSNKEC PFLHIDPESK IKDCPWYDRG FCKHGPLCRH RHTRRVICVN 150 151YLVGFCPEGP SCKFMHPRFE LPMGTTEQPP LPQQTQPPAK QSNNPPLQRS 200 201SSLIQLTSQN SSPNQQRTPQ VIGVMQSQNS SAGNRGPRPL EQVTCYKCGE 250 251KGHYANRCTK GHLAFLSGQ

The amino acid sequence of the relevant fragments of human CPSF30discussed in this patent are:

F1 Zn-Finger Domain: residues 41-59;

F2 Zn-Finger Domain: residues 68-86;

F3 Zn-Finger Domain: residues 96-114;

F4 Zn-Finger Domain: residues 124-142;

F5 Zn-Finger Domain: residues 148-166; and

F5 Zn-Finger Domain: residues 243-260.

As used herein F2F2 are residues 61-121 of SEQ ID NO.: 13:

 61 SGEKTVVCKH WLRGLCKKGD QCEFLHEYDM TKMPECYFYS 100 101KFGECSNKEC PFLHIDPESK I 121

As used herein F1F2F3 are residues 39-121 of SEQ ID NO.: 13:

 39 AV CEFFLKAACG 50  51KGGMCPFRHI SGEKTVVCKH WLRGLCKKGD QCEFLHEYDM TKMPECYFYS 100 101KFGECSNKEC PFLHIDPESK 121

The anti-viral properties of the peptides disclosed herein allow them tobe included in formulations to inhibit virus growth and proliferation.The purified anti-viral peptides may be used without furthermodifications or they may be diluted in a pharmaceutically acceptablecarrier. The invention may be administered to humans or animals,included in food and pharmaceutical preparations. They anti-viral agentsmay also be used in medicinal and pharmaceutical products (such as fluidcontainers, iv. bags, tubing, syringes, etc.), as well as in cosmeticproducts, hygienic products, cleaning products and cleaning agents, aswell as any material to which the peptides could be sprayed on oradhered to wherein the inhibition of virucidal growth on such a materialis desired.

The dosage of an anti-viral peptide necessary to prevent viral growthand proliferation depends upon a number of factors including the typesof virus that might be present, the environment into which the peptideis being introduced, and the time that the peptide is envisioned toremain in a given area.

As used herein, the phrases “pharmaceutically” or “pharmacologicallyacceptable” refer to molecular entities and compositions that do notproduce adverse, allergic, or other untoward reactions when administeredto an animal or a human. As used herein, “pharmaceutically acceptablecarrier” includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents and the like. The use of such media and agents forpharmaceutically active substances is well know in the art. Exceptinsofar as any conventional media or agent is incompatible with thevectors or cells of the present invention, its use in therapeuticcompositions is contemplated. Supplementary active ingredients also canbe incorporated into the compositions.

The active antiviral agents of the present invention may be formulatedinto classic pharmaceutical preparations and administered via any commonroute so long as the target tissue is available via that route. Theseroutes of administration include, e.g., oral, alveolar, nasal, buccal,rectal, vaginal or topical. In particular, use of the anti-viralpeptides of the present invention in a condom or diaphragm, optionallyin conjunction with a spermicidal or other contraceptive substance, isenvisioned. Alternatively, administration may be orthotopic,intradermal, subcutaneous, intramuscular, intraperitoneal orintravenous. The antiviral agent may also be administered parenterallyor intraperitoneally. Solutions of the antiviral agent may be compoundedinto a free base or pharmacologically acceptable salts can be preparedin water suitably mixed with a surfactant, such ashydroxypropylcellulose. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, and mixtures thereof and in oils. Underordinary conditions of storage and use, these preparations contain apreservative to prevent the growth of microorganisms.

The antiviral agent(s) will generally be provided in a pharmaceuticaldosage form suitable for injectable use, e.g., sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersions. For widespread use, theantiviral agents may be stable under the conditions of manufacture andstorage and must be preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. The antiviral agents willcommonly be provided with a carrier, e.g., a solvent or dispersionmedium that may include, e.g., water, ethanol, polyol (for example,glycerol, propylene glycol, and liquid polyethylene glycol, and thelike), suitable mixtures thereof, and vegetable oils. Proper dosagefluidity can be maintained, for example, by the use of a coating, suchas lecithin, by the maintenance of the required particle size in thecase of dispersion and by the use of surfactants. The prevention of theaction of microorganisms can be brought about by various antibacterialand/or antifungal agents, for example, parabens, chlorobutanol, phenol,sorbic acid, thimerosal, and the like. In many cases, the dosage formwill include isotonic agents, e.g., sugars or sodium chloride. Prolongedabsorption of the injectable compositions can be brought about by theuse in the compositions of agents delaying absorption, for example,aluminum monostearate and gelatin.

Generally, sterile injectable solutions are prepared by incorporatingthe active compounds in the required amount in the appropriate solventwith various of the other ingredients enumerated above, as required,followed by sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle that includes the basic dispersion medium and the required otheringredients from those enumerated above. Preparation of sterile powdersfor injectable solutions maybe prepared by, e.g., vacuum-drying,spray-freezing, freeze-drying or other techniques that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

As used herein, a “pharmaceutically acceptable carrier” refers tosolvents, dispersion media, coatings, antibacterial and anti-fungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

For oral administration, the antiviral agent(s) of the present inventionmay be incorporated with excipients and used in the form of ingestibleor non-ingestible mouthwashes and dentifrices. A mouthwash may beprepared incorporating the active ingredient in the required amount inan appropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the antiviral agent(s) may be incorporatedinto an antiseptic wash containing sodium borate, glycerin and potassiumbicarbonate. The antiviral agent(s) may also be dispersed indentifrices, e.g., gels, pastes, powders and slurries. The antiviralagent(s) may be added in a therapeutically effective amount to a pastedentifrice that may include water, binders, abrasives, flavoring agents,foaming agents, and humectants.

The antiviral agent(s) may be formulated in a neutral or salt form.Pharmaceutically-acceptable salts include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric and the like.Salts may also be formed with the free carboxyl groups can also bederived from inorganic bases such as, for example, sodium, potassium,ammonium, calcium, or ferric hydroxides, and such organic bases asisopropylamine, trimethylamine, histidine, procaine and the like.

Upon formulation, the antiviral agent(s) will be administered in amanner compatible with the dosage formulation and in such amount as istherapeutically effective. The formulations are easily administered in avariety of dosage forms such as injectable solutions, drug releasecapsules and the like. Sterile aqueous media that can be employed willbe known to those of skill in the art in light of the presentdisclosure, e.g., Remington: The Science and Practice of Pharmacy, 21stEdition, Lippincott Williams & Wilkins (2005), relevant portionsincorporated herein by reference. Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated for which the skilled artisan will determine the appropriatedose for the individual subject. Moreover, for human administration,preparations should meet sterility, pyrogenicity, general safety andpurity standards as required by FDA Office of Biologics standards.

In one embodiment, the vaccine comprises a pharmaceutically acceptablevehicle. The suitable vehicles may be both aqueous and non-aqueous.Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oils such as olive oil, and injectable organic esterssuch as ethyl oleate. Carriers or occlusive dressings can be used toincrease skin permeability and enhance antigen absorption. Liquid dosageforms for oral administration may generally comprise a liposome solutioncontaining the liquid dosage form. Suitable forms for suspendingliposomes include emulsions, suspensions, solutions, syrups, and elixirscontaining inert diluents commonly used in the art, such as purifiedwater. Besides the inert diluents, such compositions can also includeadjuvants, immunostimulats, immunosuppressants, wetting agents,emulsifying and suspending agents, or any combination thereof.

Examples of suitable adjuvants include, without limitation, incompleteFreund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum,Stimulon® QS-21 (Aquila Biopharmaceuticals, Inc., Framingham, Mass.),MPL® (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, Mont.),and interleukin-12 (Genetics Institute, Cambridge, Mass.).

A person of the ordinary skill in the art has a sufficient expertise todetermine the dosage of the vaccines of the instant invention. Suchdosage depends on the pathogenicity of the virus included in the vaccineand on the ability of the antigen to elicit an appropriate immuneresponse. In different embodiments of the invention, a virus vaccinecomposition of the instant invention may comprise from about 10²-10⁹plaque forming units (PFU)/ml, or any range or value therein (e.g., 10³,10⁴, 10⁵, 10⁶, 10⁷, or 10⁸) where the virus is attenuated. A vaccinecomposition comprising an inactivated virus can comprise an amount ofvirus corresponding to about 0.1 to 200 μg of the amino acid sequencesof the instant invention per ml, or any range or value therein.

The vaccines of the instant invention can be applied in multiple ways.According to one embodiment of the invention, the intranasaladministration is via the mucosal route. The intranasal administrationof the vaccine composition can be formulated, for example, in liquidform such as, for example, nose drops, spray, or suitable forinhalation. In other embodiment, the vaccine may be administered as apowder, or a cream, or an emulsion.

In another embodiment, the vaccines of the instant invention are appliedby an injection, including, without limitation, intradermal,transdermal, intramuscular, intraperitoneal and intravenous. Accordingto another embodiment of the invention, the administration is oral andthe vaccine may be presented, for example, in the form of a tablet orencased in a gelatin capsule or a microcapsule, which simplifies oralapplication. The production of these forms of administration is withinthe general knowledge of a technical expert.

As used herein, “detectable labels” refer to compounds and/or elementsthat can be detected due to their specific functional properties and/orchemical characteristics, the use of which allows the agent to whichthey are attached to be detected, and/or further quantified if desired,such as, e.g., an enzyme, an antibody, a linker, a radioisotope, anelectron dense particle, a magnetic particle or a chromophore. Thedetectable label may be half of a fluorescence resonance energy transfer(FRET) pair (for FRET variations see, e.g., U.S. Pat. No. 6,593,091,issued to Keys, relevant portions incorporated herein by reference).There are many types of detectable labels, including fluorescent labels,which are easily handled, inexpensive and nontoxic. Another example of adetection method relies on hydrogel-coated donor and acceptor beadsproviding functional groups for conjugation to biomolecules.AlphaScreen® by Berthold Technologies (Bad Wildbad, Germany) (Rouleau,N., Turcotte, S., Mondou, M. H., Roby, P. & Bosse, R. (2003) J BiomolScreen 8, 191-7.).

The present inventors recognized that certain regions of the NS1Aprotein can be targeted for the development of antiviral drugs. The NS1Aprotein is a multi-functional dimeric protein that participates in bothprotein-RNA and protein-protein interactions (Krug et al., 2003). Theeffector domain, which comprises the C-terminal two-thirds of the NS1Aprotein, contains the binding site for the 30-kDa subunit of thecellular cleavage and polyadenylation specificity factor (CPSF30)(Nemeroff et al., 1998; Li et al., 2001). This interaction results inthe inhibition of 3′ end processing of all cellular pre-mRNAs ininfected cells, and as a consequence the production of mature mRNAsencoding antiviral proteins (such as interferon, IFN) is severelyinhibited during infection (Shimizu et al., 1999; Kim et al., 2002; Noahet al., 2003). This inhibition is crucial to viral replication andspread, because influenza A virus, like several other RNA viruses(Yoneyama et al., 2004; Sumpter et al., 2005; Seth et al., 2006),efficiently activates the RIG-I RNA helicase and thereby triggers boththe activation of IRF-3 and NF-kB and the synthesis of IFN-beta andother antiviral pre-mRNAs (Geiss et al., 2002; Kim et al., 2002; Sirenet al., 2006). A recombinant influenza A/Udorn/72 virus expressing aNS1A protein with a mutated binding epitope for CPSF30 induced highlevels of IFN-beta mRNA during infection and was highly attenuated(100-1000-fold) (Noah et al., 2003; Twu et al., 2006). A 61-amino-aciddomain of CPSF30, comprising two of its zinc fingers (i.e., the F2F3fragment), was found to bind efficiently to the NS1A protein (Twu etal., 2006). The expression of the F2F3 domain in cells leads to theinhibition of influenza A virus replication and increased production ofIFN-beta mRNA, indicating that F2F3 likely blocks the binding ofendogenous CPSF30 to the NS1A protein (Twu et al., 2006). These resultsvalidate the binding epitope on NS1A for CPSF30 as a potential targetfor the development of small molecule antiviral drugs directed againstinfluenza A virus and as a target for engineering strains of virus thatcan be used as live attenuated vaccines.

Previous studies showed that residues 184-188 in the protein sequence ofthe NS1A protein are required for functional binding of CPSF30 (Li etal., 2001; Noah et al., 2003). Thus, this region is required for thebinding of CPSF30 in vitro (Li et al., 2001), and mutation of aminoacids 184-188 in the NS1A protein of a recombinant Udorn virus resultsin high attenuation coupled with increased synthesis of IFN-beta (Noahet al., 2003). These data demonstrate that this binding epitope isessential for virus replication. As shown by the structural studiesdescribed below, the 184-188 region of the NS1A protein sequenceconstitutes a subset of the CPSF30 binding epitope, observed in the 3Dstructure of the complex between NS1A effector domain and F2F3 which isdisclosed for the first time in this Disclosure.

Studies with the NS1A proteins of H5N1 viruses established that the Met(M) residue at position 106 is required for functional F2F3/CPSF30binding. We showed that the NS1A protein encoded by a pathogenic H5N1virus that was transmitted to humans in 1997 (A/HK/483/97; HK97) lacks abinding epitope for CPSF30, whereas the NS1A protein encoded by apathogenic 2004 H5N1 virus (A/Vietnam/1203/04; VN04) has acquired thisbinding epitope. We were able to generate a CPSF30 binding epitope inthe HK97 NS1A protein by changing two of its amino acids to thecorresponding amino acids in the VN04 NS1A protein, specifically bychanging I at position 106 to M and L at 103 to F. To determine theeffects of these two amino acid changes in the HK97 NS1A protein duringvirus infection, a recombinant Udorn virus was generated in which theUdorn NS gene was replaced by a HK97 NS gene encoding a NS1A proteinwith these two mutations (Ud/dmHK97 recombinant). The recombinant viruswas then compared to the recombinant Ud virus containing the wild-typeHK97 NS gene (Ud/HK97 recombinant). During multiple cycle growth therate of replication and virus yield of the Ud/dmHK97 recombinant was100-fold greater than with the Ud/HK97 recombinant virus (FIG. 1A), andis thus similar to the Udorn parent virus. During single-cycle growth,the Ud/HK97 virus induced a high level of IFN-beta mRNA production,which was almost completely suppressed in the cells infected by theUd/dmHK97 virus (FIG. 1B). Most importantly, the replication of theUd/dmHK97 virus was inhibited in F2F3-expressing cells (data not shown),indicating that the dmHK97 NS1A protein binds F2F3 in infected cells.The roles of Phe at 103 and the Met at 106 in the NS1A protein informing the F2F3/CPSF30 complex is elucidated by the herein disclosedX-ray crystal structure of the NS1A:F2F3 complex, which reveals thatamino acids 103 and 106 are required for CPSF30 binding because theirhydrophobic interactions stabilize a tetrameric structure of theNS1A:F2F3 complex (see below). It should also be emphasized thatessentially all (>98%) of the influenza A viruses (H3N2, H2N2 and H1N1)that have been isolated from humans, including the 1918 H1N1 virus, andinclude M and F at positions 106 and 103 in their NS1A proteins,respectively, indicating that these two amino acids, which are criticalfor CPSF30 binding and the resulting suppression of the production ofIFN-β mRNA, are conserved in human influenza A viruses. Accordingly, thetetrameric complex described in this disclosure is anticipated to becritical for the CSPF30 binding function of all such human influenza Aviruses.

Example 1 Process for Discovering, Designing, and Optimizing SmallMolecule Inhibitors of Influenza A Viruses, Including H5N1 Viruses

Novel Reagents and Structural Data. To further delineate the interfacebetween F2F3 of CPSF30 and the NS1A protein the inventors determined thethree-dimensional (3D) structure of the tetrameric F2F3:NS1A complex.Ninety (90) different constructs (Table 2) were screened in order tofind constructs suitable for crystallization and NMR analysis. Thesevariants, summarized in Tables 3 and 4, included 8-11 differentfull-length or subsequences of NS1A with N-terminal (N-His) andC-terminal (C-His) affinity purification tags, prepared as described inActon et al., 2005 from influenza A strains A/South Carolina/1/181918(the 1918 pandemic virusH1N1 strain), HK97 (A/HK/483/97, 1997 HongKong), VN04 (A/Vietnam/1203/04, 2005 Vietnam), and Udorn (A/Udorn/72),respectively. These were assessed in terms of expression level andsolubility (Tables 3 and 4), ability to purify, and biophysicalproperties, including 2D ¹⁵N-¹H heteronuclear single quantum coherencecorrelation spectroscopy (HSQC). Many of these reagents, disclosed forthe first time here, are useful for influenza A research purposes andfor structural studies. The inventors focused on two specific constructsthat provide particularly high expression and solubility, making themespecially useful for biochemical and biophysical studies: theC-terminal hexaHis-tagged 85-215 amino acid fragments of the NS1Aproteins from (i) influenza A/Udorn/72 (A/Udorn) and (ii) the H5N1strain A/Vietnam/1203/04 (A/VN04). These highly soluble constructs canbe produced in tens of milligram per liter quantities, and have beenproduced with SeMet labeling, for crystallography, and with¹⁵N-enrichment for NMR studies. Excellent quality HSQC NMR spectra canbe obtained for these two constructs in the pH range 5.5 to 6.5 (FIG.2). Under these conditions, these constructs are of sufficiently goodquality to indicate high feasibility for further NMR studies. They arealso useful for small molecule screening studies. In addition, the F2F3fragment of CPSF30 has been cloned into a pET expression vector with aN-terminal hexaHis tag (tag sequences are defined in Acton et al.,2005), produced with uniform ¹³C, ¹⁵N enrichment, and complete backboneand sidechain resonance assignments have been determined using standardtriple resonance NMR methods (Montelione et al., 1999). TheseNMR-optimized sample conditions and resonance assignments are useful forsmall molecule screening, lead optimization, and structure-functionstudies of F2F3:NS1A interactions.

Because virus studies have used influenza A/Udorn/72 virus to identifyCPSF30 binding activity during virus infection CPSF30 (Li et al., 2001;Noah et al., 2003), we have developed an assay for the binding of theNS1A Udorn effector domain to the F2F3 fragment of CPSF30 in vitro,using analytical gel filtration with detection of effluent bysimultaneous refractive index and static light scattering measurements.The chromatography system used for this assay is described in Acton etal., 2005, in which it is used for other applications. In these studies,illustrated in FIG. 3, the NS1A Udorn (85-215) construct elutes as adimer, with molecular weight of ˜30 kDa. When this effector domainconstruct is first mixed with the 8.5 kDa F3F3 fragment, the resultingcomplex elutes with a molecular weight of ˜50 kDa, indicating astoichiometry of 2 F2F3:2 NS1A effector domains. This gel filtrationassay provides a convenient qualitative measure of F2F3 binding by NS1Aeffector domains.

Suitable samples of NS1A Udorn (85-215); the NS1A-Udorn (85-215):F2F3complex, and other constructs listed in Table 2 and their complexes withF2F3 and CPSF30-derived constructs, can be produced and assessed usingthis gel filtration assay. These can then be crystallized for X-raycrystallographic analysis. Initial crystals of the NS1A-Udorn(85-215):F2F3 complex were obtained by high-throughput robotic screeningusing service facilities available through the Hauptman Woodward Res.Inst. (Buffalo N.Y.) (Luft et al., 2003; Acton et al., 2005).Crystallization optimization was then carried out for the NS1A-Udorn(85-215):F2F3 complex at Rutgers, yielding diffraction qualitybi-pyramid crystal of size 0.15×0.15×0.25 mm³. Conditions providingcrystals, and a description of these crystals, are presented hereinbelow. Three-wavelength Se-Met multiple anomalous diffraction (MAD) (2.3Å resolution) and a complete native (1.95 Å resolution) data sets werecollected at X25 beam line at Brookhaven National Laboratories. Thestructure was solved by MAD techniques (Hendrickson, 1991) and refinedto 1.95 Å resolution, and is currently refined to a R factor of 0.225and R free of 0.245, respectively.

Crystallization of NS1A Udorn (85-215):F2F3 Complex. Initialcrystallization conditions for NS1A Udorn (85-215):F2F3 complex wereobtained by using robotic crystallization facility at the Buffalofacility described above. The most promising condition use 1.77 Mpotassium nitrate (KNO₃) at pH 5.0 as precipitant. This initialcrystallization condition was optimized manually at Rutgers. Goodquality crystals were obtained using 0.25-1.0 M KNO₃ and 10-20%sucrose/glucose as precipitant at pH 5-6. Diffraction quality crystalswere obtained when P94S mutant F2F3 was used in the complex, thoughsimilar conditions are expected to provide crystals of the complex withthe wild-type (P94) F2F3 sequence. The NS1A Udorn (85-215):F2F3 complexwas crystallized with the symmetry of the space group P4₁ with cellparameters a=b=50.96, c=205.39 Å and α=β=γ=90°. A variant used in theX-ray crystal structure determination of NS1 (85-215):F2F3 complex hasSer in place of Pro at position 94.

FIGS. 4A and 4B disclose the crystal structure of the Udorn NS1Aeffector domain (residues 85-215) bound to the F2F3 fragment of CPSF30.Consistent with gel filtration data, the complex forms a tetramericcomplex, in which two F2F3 molecules bridge two NS1A effector domains.The structure reveals for the first time the novel spatial arrangementof the subunits in the 2 NS1A: 2 F2F3 complex. This tetramericarrangement was completely unexpected and novel.

Key residues Met106/Met106′ of NS1A are packed in the core of thetetrameric interface or “tetramerization epitope,” explaining itscritical role in CPSF30 binding, as discussed above. However, while Met106/Met106′ are at the core of the tetrameric complex, they are not partof the primary F2F3/CPSF30 binding pocket. FIG. 5 illustrates the F2F3binding pocket on the surface the Udorn NS1A effector domain. Specificinteractions in this primary binding pocket involve residues K110, I117,I119, Q121, V180, G183, G184, and W187 of NS1A and residues F97, F98,F103, and K110 of the F3 finger of CPSF30. It should be noted that theseresidues include not only amino acids in, or adjacent to, the 184-188sequence previously identified as part of the CPSF30 binding epitope bymutagenesis studies, but also amino acids in other regions in the NS1Aprotein not previously recognized to be essential for CPSF30 binding.Mutation of residues G183 or G184 in NS1A (85-215) each to Arg or Aspdisrupts or reduces F2F2 binding affinity and/or tetramer formation,based on analytical gel filtration measurements without significantlydisrupting the native structure of NS1A (85-215) indicated by ¹⁵N-¹HHSQC NMR spectra (data not shown), demonstrating a functional role ofthese sites within the CPSF30 binding epitope of NS1A. Recombinant Udornviruses encoding NS1A proteins in which either G184 or W187 was changedto Arg are attenuated (data not shown), further demonstrating that theseamino acids are required for virus replication. The eight residues inthe CPSF30-binding epitope of NS1A (FIG. 5) are almost 100% conservedamongst influenza A viruses isolated from humans (Macken et al., 2001),except for Ile117 which is frequently replaced by Met. Based on thisstructure, disclosed for the first time here, we propose disruption of2NS1A:2F2F3 tetramerization as a strategy to inhibit influenza A virusin human. Based on our structural data, we further propose that a smallmolecule inhibitor that binds tightly to the above described conservedepitope and surrounding regions will disrupt CPSF30 binding and willinhibit influenza A virus in humans. This pocket on the surface of theNS1A effector domain is the target of our proposed drug discoveryprocess.

Table 1 describes some of the specific location of the epitopesdescribed herein as the tetramerization, RNA binding and CPSF30 bindingepitopes of NS1A.

TABLE 1 Tetramerization, RNA binding and CPSF30 binding epitopes ofNS1A. NS1A Interacting amino amino acids of acids F2F3 Remark F103 L72,Y88, M93, F103 contributes to both the tetramer interface and theCPSF30-binding epitope P111 F103 interacts with F2 and F3 domains ofCPSF30, may play a role in defining angle between F2 and F3appropriately; mutation of 103 could affect an intermolecular saltbridge between R73 and D125. L105 P111, F112, L105 contributes to boththe tetramer interface and the CPSF30-binding epitope M124 (NS1A-2)Adjacent to F103 and also interacting at the tetramer interface with a2^(nd) NS1A molecule. M106 N107 M106 contributes to both the tetramerinterface and the CPSF30-binding epitope. M106I mutation would effectthe positioning of N107 (of F2F3) and/or Q121 (NS1A same molecule)involved in F2F3-NS1A H-bonds. K110 K110 K110 contributes to theCPSF30-binding epitope. K110 has H-bond with the main-chain carbonyl ofK101 of F2F3. I117 F102 I117 contributes to the CPSF30-binding epitope.Part of the hydrophobic pocket that binds F3 I119 F98, F102 I119contributes to the CPSF30-binding epitope. Part of the hydrophobicpocket that binds F3 Q121 S105, S106 Q121 contributes to theCPSF30-binding epitope. H-bond with amino group of S106 and Oγ of 105(both from CPSF30) D125 R73 D125 contributes to the CPSF30-bindingepitope. Salt bridge with R73 (of CPSF30) L144 No inter- L144 is buriedinto the hydrophobic core of NS1A that probably defines the foldmolecular of the protein. A mutation of L144 may alter the shape ofNS1A, resulting in interaction reduced CPSF30 and/or dsRNA bindingactivities V180 Y97, K101, V180 contributes to the CPSF30-bindingepitope. Part of the F3-binding pocket. F102 G183 Y97 G183 contributesto the CPSF30-binding epitope. Part of a helix that forms a wall of theF3-binding pocket. A side-chain may affect (enhance or disrupt) thebinding of F3. G184 Y97, F98 G184 contributes to the CPSF30-bindingepitope. Part of a helix that forms a wall of the F3-binding pocket.Positioned between Y97 and F98 side chains. A mutation of G184 wouldalter the shape of the pocket and can severely affect the binding of F3.W187 C96, Y97, F98, W187 contributes to the CPSF30-binding epitope.Participates in the pocket F112 formation.

Recently, the crystal structure of the NS1A effector domain (residues 79to 205) of the influenza A/PR8/34 (PR8) virus strain was reported(Bornholdt and Prasad, 2006). This PR8 influenza strain is adapted forpassage in mouse, presumably as a result of mouse adaptation, the PR8 NSgene attenuates virus growth in mammalian cells in tissue culture (Ozakiet al, 2004). The PR8 NS1A protein does not bind F2F3 because it lacksthe consensus human sequence at positions 106 (I instead of M) and 103(S instead of F) (Macken et al, 2001), therefore we can determine if theattenuation of growth in mammalian cells is caused primarily by thechanged amino acids at positions 106 and 103 in the PR8 NS1A protein(data not shown). The absence of a CPSF30 binding epitope in the PR8NS1A protein presumably resulted from the large number of passages inmice that have adapted the virus for replication in mice. Consequently,the 3D structure of effector domain of the PR8 NS1A protein (Bornholdtand Prasad, 2006) is not suitable for structure-based drug discoveryefforts. In addition, its structure may differ from that of a NS1Aprotein containing a functional CPSF30 binding epitope. Indeed, the 3Dstructures of the Udorn effector domain monomers in our complex aresimilar to, but not identical with, the structure of the monomers of thePR8 NS1A effector domain structure (FIG. 6). The PR8 NS1A structure doesnot have a F2F3-binding epitope like that observed in the 3D srructureof the Udorn NS1A structure described herein. In particular, the PR8NS1A structures lack the hydrophobic pocket that binds F2F3 in theNS1A:F2F3 complex. Moreover, the dimeric interface in crystal structureof the PR8 NS1A effector domain has an intermolecular beta-sheetinteraction (FIG. 7) that is completely different from the interface inthe crystal structure of the Udorn NS1A effector domain bound to F2F3.Therefore, the dimer of the effector domain of the PR8 NS1A protein isnot relevant for the binding of CPSF 30 and to our proposed drug-designstrategy.

Fluorescence polarization (FP) assay. Fluorescence polarization (FP) isa spectroscopic method that measures the rotational rate of a sample insolution. It applies polarized light to excite a fluorophore andmeasures the polarization characteristics of the emitted light (Nasirand Jolley, 1999; Roehrl et al., 2004b). If the sample tumbles slowlycompared to the lifetime of the fluorescence, the emitted light retainssome of the incident polarization. However, if the sample tumbles fast,the emitted light is isotropic. Therefore, the degree of anisotropy(polarization) of the fluorescent light emitted from a sample provides ameasure of the fluorophore's rotational correlation time in the boundstate. Given appropriate bound-state lifetimes, rotational correlationtimes, and changes in rotational correlation upon complex formation, FPassays can be used to measure binding affinities and to screen forbinding inhibitors (Schade et al., 1996; Seethala and Menzel, 1998;Roehrl et al., 2004a).

FP with fluorescently-labeled F2F3. A FP assay of F2F3 binding by NS1Ais being implemented. As mentioned above, complex formation between F2F3and various effector domain constructs is observed in gel filtrationstudies (see FIG. 3), indicating a dissociation constant K_(d) tighterthan low micromolar. These data of FIG. 3 demonstrate the feasibility ofimplementing a FP assay for detecting NS1A:F2F3 binding. Moreover,excellent HSQC spectra and essentially complete NMR resonanceassignments are available for the F2F3 construct (data not shown); thesewill be used to assess the native structural integrity offluorescently-labeled CPSF30 fragments.

In one embodiment of the invention, we will generate a fluorescein (orother fluorophore)—labeled F2F3 (Fluo-F2F3) substrate for FP assay. TheF2F3 sequence of human CPSF30 contains 7 Lys and 6 Cys residues, andfluorophore labeling requires careful consideration to ensure properfolding with Zn ligation by these six Cys residues. Two primarystrategies will be pursued for labeling F2F3: (i) chemical synthesiswith a fluorophore label and (ii) synthesis or biosynthesis of an analogsuitable for specific labeling. The 61-residue F2F3 is sufficientlyshort to allow fluorophore labeling by solid phase peptide synthesis(for example, by a commercial supplier, such as New England Peptide,Inc. Gardner, Mass.). The crystal structure of F2F3 bound to the NS1Aeffector domain indicates that the N-terminal region is not involved inkey binding interactions, and suggests that it may be possible togenerate shorter constructs with similar binding affinities. Thoseconstructs that show tight binding can be synthesized in 100 mg amountsand assayed for structural integrity by ¹⁵N-1H HSQC NMR analysis atnatural isotopic abundance using a 800 MHz NMR system with cryoprobe.

If for some reason this approach is not successful, fluorophone-labeledF2F3 will be produced by overexpression with a ‘flash’ peptide sequencetag (Griffin et al., 1998; Griffin et al., 2000; Abrams, 2002). When thetetra-cysteine tag [NH₂-Cys-Cys-Pro-Gly-Cys-Cys . . . ] is incorporatedinto proteins/peptides it can be specifically conjugated to abiarsenyl-fluorophore ligand (sold by Invitrogen as theirLumio-technology). The modification can even specifically label thetetra-cysteine tag in vivo (Griffin et al., 2000), and thus should notreact with any of the six cysteine residues on F2F3. If even both ofthese approaches prove to be problematic for this tandem Zn-fingerdomain, alternative strategies for fluorophore labeling are available,such as the method of Ting and colleagues (Chen and Ting, 2005) thatutilizes biotin ligase together with appropriate cofactors to introducefluorescein in a short N-terminal acceptor peptide lacking any Cysresidues, allowing the fluorophore to be introduced subsequent toforming the Zn fingers.

Using any of the above labeling approaches, or other possibleapproaches, the labeled F2F3 can then be folded with Zn ligation usingthe same protocol we have developed for unlabeled F2F3. In exploringeach of these approaches, we will verify that the fluorophore in theN-terminal tag becomes sufficiently immobilized in the complex toprovide a significant change in fluorescence anisotropy. If required, amore immobilized Cys residue within the F2F3 sequence with be introducedwithout interfering with Zn finger formation, using our 3D structure ofthe NS1A (85-215):F2F3 complex as a guide. Using one of these labelingroutes, or any approach for introducing the fluorescence tag, theresulting FP assay will be validated by measuring competition withunlabeled F2F3.

The crystal structure of the complex between NS1A Udorn (85-215) and theF2F3 CPSF30 reveals that most of the intermolecular interactions betweenNS1A and F2F3 involve the F3 Zn-finger domain (FIG. 5). Accordingly, asmaller flourescein-labeled F3 finger (Fluo-F3), with ˜4 kDa molecularweight, may have preferential properties in developing a FP assay.Though the binding of the NS1A effector domain to Fluo-F3 is likely tobe somewhat weaker than the binding to F2F3, the change in rotationalcorrelation time, and thus the signal-to-noise of the FP assay, will besignificantly higher using Fluo-F3 than using Fluo-F2F3. These designprinciples may be advantageous in development of a high throughput FPassay useful in screening for inhibitors of CPSF30 (or variants,mutants, or fragments thereof) binding to NS1A (or variants, mutants, orfragments thereof).

The crystal structure of the complex between NS1A Udorn (85-215) and theF2F3 fragment further suggests that a tandem repeat of the F2F3fragment, connected by a short flexible linker, F2F3-F2F3, could alsobind in a manner similar to the two independent F2F3 molecules, thoughpotentially with much tighter binding affinity. Accordingly, we willgenerate a variety of F2F3-F2F3 constructs, as outlined below, assaytheir binding, and assess the oligomerization states of these complexesby gel filtration with light scattering detection. If indeed a tandemrepeat F2F3-F2F3 construct can be generated with even tighter bindingthan F2F3 itself, it will be used in a more sensitive FP assay, capableof detecting only more tightly-binding inhibitors.

The magnitude of the change in fluorescence anisotropy, which determinesthe signal-to-noise of the FP assay, depends on the change in rotationalcorrelation time of the fluorophore upon complex formation. If requiredto enhance this change in rotational correlation time, the relative sizeof the NS1A component of NS1A:CSPF30 (or fragments thereof) complexesdescribed above can be further increased by expression as a largerfusion protein (e.g., fused to maltose binding protein), or byattachment to beads or microscopic particles using, for example, alreadyavailable constructs of Tables 2, 3, and 4 with either N- or C-terminalhexHis tags, or using other affinity tags.

Example 2 Constructs to be Used in Developing Fluorescence PolarizationAssay

The crystal structure of the complex between NS1A Udorn (85-215) and theF2F3 fragment was examined to identify potential linking sites so thatthe C-terminus of one segment of F2F3 (or portion thereof) would have afavorable path for linking to the N-terminus of the second segment.Considerations for favorable linking geometry include the distancebetween termini to be linked, the path the linker would need to take,and linker composition and length. A common peptide linker compositionused for connecting domains in engineered proteins is some combinationof Gly and Ser residues, allowing for conformational flexibility andsolubility while remaining somewhat neutral in chemical content, e.g.-Gly-Ser-, or -Gly-Ser-Gly, etc.

Constructs were designed based on the crystal structure of the influenzavirus NS1A-CTD protein complex with the F2F3 fragment from the humanCPSF30 protein, disclosed for this first time in this Disclosure. Theexpectation is that a single-chain construct of the F2F3 binding regionsobserved in the crystal structure would bind more tightly to the NS1Adimer than the separate domains, and might have significant antiviralactivity.

The structure was examined to identify potential linking sites so thatthe C-terminus of one segment of F2F3 (or portion thereof) would have afavorable path for linking to the N-terminus of the second segment.Considerations for favorable linking geometry include the distancebetween termini to be linked, the path the linker would need to take,and linker composition and length. A common peptide linker compositionused for connecting domains in engineered proteins is some combinationof Gly and Ser residues, allowing for conformational flexibility andsolubility while remaining somewhat neutral in chemical content, e.g.-Gly-Ser-, or -Gly-Ser-Gly, etc.

The sequence of CPSF30 F2F3 used for the specific designs is that usedin the crystal structure determination, but amino acid replacements canbe considered. The N-terminal portion might include a hexa-His or othertag for convenient purification; alternatively a tag might be encoded atthe C-terminus of the construct. Other purification tags may bepreferable to oligo-His owing to potential interference with thechelation of zinc ions by the F2 and F3 “Zn finger” segments, which isrequired for the integrity of the three-dimensional structure and foreffective binding to NS1A. In all of these F2F3 sequences, residue Pro92may be substituted by Ser.

The constructs involving single F3 domains (“one finger”) as opposed toF2F3 domains (“two fingers”) are potentially valuable asminimal/miniature versions because the interaction between separate F2F3chains in the dimer (chains F2F3-A and F2F3-B) is mediated entirely byresidues in F3 and does not involve any residues from F2. The F3 domainitself, or variants, could be used as an antiviral drug.

Relatively few of the residues in F2 make important interactions withthe NS1A protein in the dimer of dimers; notable exceptions are Leu72and Arg73, whose side chains interact extensively with NS1A. The Leu72and Arg73 interactions could be maintained by a strategic linkage of aLeu-Arg or Arg-Leu peptide to Thr91 (proposed N-terminus of the F3domain construct). L72-T91 distance is 14.2 Å; R73-T91 distance is 17.6Å. Possible extensions to the one-finger F3 domain construct would beLeu-Arg-GSGSG-F3 or Arg-Leu-GSGSG-F3 (see proposed constructs 5a and5b).

F2F3 sequence (numbered as in human CPSF30 protein):

(SEQ ID NO.: 2) 61           71         81         91M SGEKTVVCKH WLRGLCKKGD QCEFLHEYDM TKMPECYFYS 101        111KFGECSNKEC PFLHIDPESK I

Construct 1. F3-F3: Single F3-linker-F3 construct (tandem F3construct)(abbreviated: (91-116)-linker(11+)-(91-116)). Distance fromF2F3-A Asp116 Ca to F2F3-B Thr91 Ca is 33.3 Å; linker can be 10+flexible amino acids;

(SEQ ID NO.: 3) 11: -GSGSGSGSGSG- also written as -(GS)₅G-;(SEQ ID NO.: 4) 13: -GSGSGSGSGSGSG- also written as -(GS)₆G-;(SEQ ID NO.: 5) 91         101        111    linker 91TKMPECYFYS KFGECSNKEC PFLHID-(GS)_(N)G-TKMPECYFYS 101        111 116KFGECSNKEC PFLHID

C-terminal residue of tandem construct can be D116 or any extension upto 1121; crystal structure extends only to P117 in one copy and E118 inthe other. Both N- and C-termini can have extensions including hexaHisand other tags for convenient purification as discussed in the text.

Construct 2. F2F3-F3: Same as previous construct except that theN-terminal portion of the construct encompasses entire F2F3 segment(abbreviated) (60-116)-linker(11+)-(91-116)

(SEQ ID NO.: 6) 61           71         81         91M SGEKTVVCKH WLRGLCKKGD QCEFLHEYDM TKMPECYFYS101        111    linker 91         101KFGECSNKEC PFLHID-(GS)_(N)G-TKMPECYFYS KFGECSNKEC 111 116 PFLHID

Construct 3. F2F3-F2F3: F2F3-linker-F2F3 construct (tandem F2F3construct)(abbreviated: (60-116)-linker(15+)-(62-116)). Distance fromF2F3-A Asp116 Ca to F2F3-B Gly62 Ca is 51.1 Å; linker should be 14+flexible amino acids.

(SEQ ID NO.: 7) 15: -GSGSGSGSGSGSGSG- also written as -(GS)₇G-;(SEQ ID NO.: 8) 17: -GSGSGSGSGSGSGSGSG- also written as -(GS)₈G-;(SEQ ID NO: 9) 61    71           81      91         101M SGEKTVVCKH WLRGLCKKGD QCEFLHEYDM TKMPECYFYS    111           linker 62        71KFGECSNKEC PFLHID-(GS)_(N)G-GEKTVVCKH WLRGLCKKGD81         91        101         111QCEFLHEYDM TKMPECYFYS KFGECSNKEC PFLHID.

Construct 4. F3-F2F3: F3-linker-F2F3 construct (abbreviated:(91-116)-linker(15+)-(62-116)). Distance from F2F3-A Asp116 Ca to F2F3-BGly62 Ca is 51.1 Å; linker should be 14+ flexible amino acids.

(SEQ ID NO.: 10) 91         101        111 116 linker 62TKMPECYFYS KFGECSNKEC PFLHID-(GS)_(N)G-GEKTVVCKH71         81         91         101        111WLRGLCKKGD QCEFLHEYDM TKMPECYFYS KFGECSNKEC PFLHID

Construct 5. XF3-F3: Extended F3-linker-F3 construct. Linker 1 will linka Leu-Arg or Arg-Leu dipeptide to the one-finger F3 domain in a mode toincorporate the important L72 and R73 side-chain interactions from F2that are otherwise missing. Linker 2 can be 10+ flexible amino acids:

Construct 5a (abbreviated:Leu-Arg-linker(5+)-(91-116)-linker(11+)-(91-116)):

(SEQ ID NO.: 11) 72  73  linker 1 91       101       111 116Leu-Arg-GSGSG-TKMPECYFYS KFGECSNKEC PFLHID-linker 2 91       101        111 116(GS)_(N)G-TKMPECYFYS KFGECSNKEC PFLHID

Construct 5b (abbreviated:Arg-Leu-linker(5+)-(91-116)-linker(11+)-(91-116)):

(SEQ ID NO.: 12) 73  72 linker 1 91      101         111 116Arg-Leu-GSGSG-TKMPECYFYS KFGECSNKEC PFLHID-linker 2 91       101        111 116(GS)_(N)G-TKMPECYFYS KFGECSNKEC PFLHID

Each of the constructs 1-5b above, and variants thereof, will inhibitCPSF30 binding by NS1A, and can potentially be used as lead compoundsfor drug development and/or as antiviral drugs useful in preventing ortreating influenza A infection in humans, poultry (e.g. chicken), andother animals.

Structure-based Lead Optimization using X-ray Crystallography and NMR.The sample preparation and structural characterization of NS1A effectordomains from multiple influenza strains, allow the use of both NMR andX-ray crystallography in hit validation, lead compound optimization, anddrug discovery.

Crystallography for lead optimization. X-ray analysis has historicallybeen very important to structure-based inhibitor design. Compoundsidentified as inhibitor “hits” by kinetic assays can be used to formcomplexes with the target protein. X-ray analysis will reveal the modeof inhibitor binding and act as a basis for further inhibitor design.Crystal•inhibitor complexes can also be formed by soaking inhibitorsinto preformed crystals, or complexes may be grown byco-crystallization. The latter case may be necessary if the complextriggers conformational changes. In either case, diffraction data iscollected on a rotating anode source, processed, and converted toelectron density maps. The orientation and occupancy of the inhibitorswill be observed and compared with binding modes predicted from virtualscreening or models from earlier design cycles. In this way, inhibitorsanalyzed in one cycle of design will feed into subsequent rounds ofstructural analysis and re-design in the general process ofstructure-based drug design (Kuhn 2002; Scapin 2006)

Cocrystallization and Crystal soaks. Using crystallization conditionsthat we have already optimized for the NS1A effector domains, describedabove, cocrystallizations and crystal soaks can be carried out with leadcompounds identified in our high throughput screening efforts. Trialswill be carried out to crystallize NS1A effector domain:lead compoundcomplexes. Diffraction data can be collected using either home orsynchrotron X-ray sources and the resulting difference electron densitymaps and 3D structures can be used to characterize small moleculebinding epitopes and bound-state conformations. These data can be usedscreening for lead compounds either one at a time or in high throughputformat, lead compound validation, and optimization of lead compounds,using methods familiar to a person skilled in the art.

NMR for lead identification optimization. NMR is also a powerful methodfor validating intermolecular interactions, and is used extensively incommercial pharmaceutical drug discovery efforts to identify leadcompounds and their protein binding sites (Shuker et al., 1996; Hajduket al., 1997; Moore, 1999a, 1999b; Muegge et al., 1999; Moy et al.,2001; Powers, 2002; Lepre et al., 2004; Rush and Powers, 2004; Mercieret al., 2006; Petros et al., 2006). NMR chemical shift perturbations, ofeither the protein or the ligand, can be used for screening for smallmolecule lead compounds, validating initial small molecule hitsidentified with htp or virtual screening assays, locating thecorresponding binding site in the 3D protein structure, and to guiderational lead optimization. NMR methods uniquely complementcrystallography studies. Strengths of NMR analysis methods include (i)the ability to detect even weak (K_(d) up to ˜500 micromolar) bindinginteractions, (ii) compatibility with systems that undergo structuralchanges upon ligand binding which may not be accommodated by acrystalline lattice, (iii) the ability to adjust solvent conditions overa wide range, and (iv) high sensitivity and very low samplerequirements.

Using NS1A effector domain constructs that have already been shown toprovide high quality HSQC spectra (e.g., FIG. 2), NMR chemical shiftperturbation can be used to validate initial small molecule hitsidentified with high-throughput (htp) or virtual screening assays,locating the corresponding binding site in the 3D protein structure, andfor rational lead optimization. Hits identified in htp FP assays will becharacterized by assessing perturbations in HSQC spectra for whichresonance assignments will be available, and interpreting these effectson the available 3D structures of NS1 effector domains. Some methodssuitable for lead compound screening, identification, and optimizationenvisioned for use with our invention are summarized in C. A. Lepre, J.M, Moore, and J. W. Peng, Theory and Applications of NMR-based Screeningin Pharmaceutical Research (Chem. Rev. 104, 3641-3675 (2004)), which isincorporated herein by reference. The NMR infrastructure required forsuch work are available at most large research institutions, includingby not limited to Rutgers University, and include 5 mm 600 and 800 MHzNMR cryoprobe systems, as well as a 1 mm 600 MHz NMR equipped with anautomated sample changer. The cryoprobes enable recording ofhigh-quality HSQC data using 150 microliter samples at proteinconcentrations as low as 10 micromolar, and the 1 mm NMR probe provideshigh quality HSQC data on 7 microliter samples at 100 micromolarconcentrations. Such NMR technologies allow NMR screening for leadcompound optimization with as little as ˜500 nanomoles of each sample.Where appropriate, the 3D structures of tightly bound small moleculelead compounds may also be determined by NMR methods if they cannot besolved by crystallographic methods. These data will be used in redesignand lead optimization as outlined for crystallographic data above.

Expressed soluble constructs of NS1A outlined in Tables 2, 3 and 4, ortheir variants, can also be used to screen for small molecule leadcompounds using NMR detection of resonances of the small molecule, usinglimits described by (but not limited to) C. A. Lepre, J. M, Moore, andJ. W. Peng, Theory and Applications of NMR-based Screening inPharmaceutical Research (Chem. Rev. 104, 3641-3675 (2004)).

Where appropriate, the 3D structures of tightly bound small moleculelead compounds may also be determined by NMR methods if they cannot besolved by crystallographic methods. These data will be used in redesignand lead optimization using synthetic chemistry, as outlined forcrystallographic data above.

Antiviral assays. Having identified inhibitors of the interactionbetween NS1A and CPSF30, the inhibitors would be tested for theirability to inhibit influenza A virus replication in tissue culturestudies. Plaque reduction assays in MDCK cells will be used to assaycompounds for their ability to inhibit influenza A virus replication(Twu et al., 2006). Monolayers of MDCK cells will be infected withapproximately 100 plaque-forming units (PFU) of influenza A/Udorn/72virus, and after virus adsorption the cells will be overlaid with agarcontaining a concentration of a compound ranging from 0.001micrograms/ml to 10 micrograms/ml. Plaques will be counted in duplicateplates and compared to the number of plaques in controls not exposed tothe chemical. In parallel cytotoxicity evaluations of lead compoundswould be carried out on MDCK cells using the Roche reagent WST-1(Berridge et al., 2005). The rate of WST-1 cleavage by mitochondrialdehydrogenases, yielding a product with absorbance at 450 nm, correlateswith the number of viable cells. These assays will be carried out in96-well tissue culture plates, and cell viability will be assayed bydetermining absorbance at 450 nm using an ELISA plate reader. Once themost promising lead compounds with low cytotoxicity in both MDCK andhuman (A549) cells have been identified, we will determine the effect ofseveral concentrations of the compounds on the rate and extent of virusreplication during multiple-cycle growth in MDCK cells. The compoundswith the greatest inhibitory activity at the lowest concentrations willbe used for subsequent studies in a suitable animal model (ferrets).

Applications of NS1A:CPSF30 Inhibitors. These inhibitors can bedeveloped into antiviral drugs that will be effective in the control ofinfluenza virus epidemics and pandemics in humans. Because there is thepotential for such epidemics to be man-made, i.e., using influenza Avirus as a bioterrorist weapon, such antivirals would also be crucial inthis situation. Influenza antiviral drugs will also be important for thecontrol of influenza A infections in commercial poultry stocks, such aschickens.

TABLE 2 pET-based vectors for NS1A proteins and fragments from variousflu strains. 1918 HK97 VN04 Udorn Full-length N-His and C-His N-His andC-His N-His and C-His N-His and C-His  1-215 N-His and C-His N-His andC-His N-His and C-His N-His and C-His 73-215 N-His and C-His N-His andC-His N-His and C-His N-His and C-His 85-215 N-His and C-His N-His andC-His N-His and C-His N-His and C-His 91-215 C-His Not attempted C-HisC-His  1-211 N-His and C-His N-His and C-His N-His and C-His N-His andC-His 85-211 N-His and C-His N-His and C-His N-His and C-His N-His andC-His 73-211 N-His and C-His N-His and C-His N-His and C-His N-His andC-His 91-211 C-His Not attempted Not attempted Not attempted 85-204C-His C-His C-His C-His 91-204 C-His Not attempted C-His C-His

TABLE 3 N-terminal His tag NS1 Expression/Solubility results for NS1Aproteins and fragments from various flu strains 1918 HK97 VN04 Udorn FLyes/low yes/low no/na yes/low  1-215 yes/low yes/low yes/no yes/low73-215 yes/no yes/med yes/no yes/low 85-215 yes/low yes/low yes/lowyes/med  1-211 yes/no yes/low yes/no yes/low 85-211 yes/low yes/noyes/med yes/med 73-211 yes/low yes/no yes/med yes/low

TABLE 4 C-terminal His tag NS1 Expression/Solubility results for NS1Aproteins and fragments from various flu strains 1918 HK97 VN04 Udorn FLyes/low yes/no no/na yes/low  1-215 yes/no yes/medium yes/no yes/no73-215 na na yes/no yes/low 85-215 yes/medium yes/good yes/low yes/low91-215 yes/low yes/low na yes/low  1-211 no/na na yes/no na 85-211yes/no yes/no yes/low yes/no 73-211 na yes/no yes/good yes/no 91-211yes/no na na na 85-204 yes/low yes/low yes/low yes/low 91-204 yes/noyes/no na yes/no

Creating Attenuated Influenza Virus Strains Suitable for Avian and HumanInfluenza Vaccine Development. The structural information disclosed herecan be combined with site-directed mutagenesis data for NS1A to engineerattenuated influenza A virus strains suitable for use as live attenuatedvaccines in humans and livestock. Multiple, weakly attenuating mutationswill be created in the CPSF30 and dsRNA-binding epitopes to generate NS1proteins with reduced binding affinity in vitro. The effects of thesemutations on the structural integrity of NS1A variants can be assessedwith HSQC NMR data and/or crystallization and X-ray crystallography.These data will then be used to engineer viral strains of influenza Awith various degrees of attenuation. These viral strains will beengineered to contain multiple base changes to minimize the potential ofthe virus to revert to virulence. Such influenza A viruses would beattenuated in normal cells, and in humans and animals, but would not beattenuated in cells lacking interferon genes (e.g. cell culture Verocells), thereby enabling the production of large amounts of theseattenuated viruses suitable for use as a live virus vaccine.

Methods for development of attenuated flu vaccines are described in U.S.Provisional Patent Application Ser. No. 60/737,742, “Novel Compositionsand Vaccines Against Influenza and Influenza B Infections,”, and PTCpatent application Filed Nov. 17, 2006 “Novel Compositions and VaccinesAgainst Influenza and Influenza B Infections,” relevant portions andtables incorporated herein by reference, relevant portions incorporatedherein by reference.

A person of ordinary skill in the art will understand that because ofthe degeneracy of the genetic code, a large number of nucleic acidsequences can be generated in accordance with this invention. Theproduction of the viruses of the instant invention (or the respectiverecombinant NS1A protein or respective NS1B protein comprising saidamino acid sequences) can be achieved by recombinant DNA technology.Nucleic acid sequences encoding the amino acid sequences of the instantinvention can be produced using methods well known in the art,including, for example, chemical synthesis, PCR and site-directedmutagenesis.

The instant invention provides recombinant influenza A virus comprisingthe amino acid sequences which are at least 70% identical but less than100% identical either to dsRNA binding domains of NS1A. These virusesmay be generated, for example, by reverse genetic system, wherebyinfluenza virus can be generated by transfection of multiple DNAswithout a helper virus. This technique was described in Fodor et al.,1999. Essentially, in that study the technique involved transfectinginto a host cell a combination of plasmids containing cDNAs for theviral RNA segments (including NS1 proteins), proteins of viral RNAdependent polymerase complex (PB1, PB2, and PA), and nucleoprotein.Further, it is possible to transfect host cells (such as, for example293 cells or Vero cells) with a plasmid encoding a recombinant NS1Aprotein (for rescuing influenza A viral phenotype) containing theappropriate amino acid sequence of the instant invention. The resultingviruses may be used for creation of vaccines, as described below

In another embodiment, the virus can be propagated in suitable host,such as, for example, chicken eggs, without a need for transforming ahost cell with multiple plasmids. In this embodiment, essentially,clinical isolates of human influenza virus are taken from infectedpatients and are reassorted in embryonated chicken eggs withlaboratory-adapted master strains of high-growth donor viruses.

To prepare the recombinant NS1A viruses for different purposes(including, without limitations, the use of the virus as a vaccine or apart thereof), each of these mutations may be carried out individuallyon constructs of NS1A suitable for biochemical characterization,possibly including but not limited to the constructs outlined in Tables3 and 4. Other variants of NS1A or affinity tags (e.g. FLAG tags) mayalso be used. These proteins may be purified and characterized withrespect to structural integrity by comparing the circular dichroismand/or NMR spectrum and/or X-ray crystal structures with those of thecorresponding wild-type NS1 construct. The several constructs may thenbe assayed for dsRNA binding as described elsewhere (Chien et al.,2004), or using other methods of assessing protein-dsRNA bindingaffinities commonly used for such studies, such as, for example,sedimentation equilibrium, gel electrophoresis, or gel filtrationchromatography. These data will be used to assess the effect of singlesite mutants at these sites revealed by the structural models to beimportant for dsRNA recognition. Further, sets of double, triple, andquadruple mutants of residues in the CPSF30 binding epitope, tetramerinterface, dsRNA binding epitope, and combinations thereof would also begenerated in these NS1A constructs and assessed for structural integrityand binding activities in vitro. This experimental design will allow aperson of the ordinary skill in the art to identify mutant forms of NS1Aand NS1A varients with minimal structural disruption but with reducedCPSF30 and/or dsRNA binding affinity.

The invention encompasses methods of selecting viruses which have thedesired phenotype, i.e., viruses which have low or no dsRNA bindingactivity, whether obtained from natural variants, spontaneous variants(i.e., variants which evolve during virus propagation), mutagenizednatural variants, reassortants and/or genetically engineered viruses.Such viruses can be best screened in differential growth assays thatcompare growth in host systems which have attenuated and normal immuneresponse to influenza A viruses. Viruses which demonstrate better growthin the hosts having the attenuated response versus the normal responseare selected; preferably, viruses which grow to titers at least one loggreater in the host systems with the attenuated response as compared tothe host system with the normal response are selected.

The present invention also encompasses methods of growing and isolatingmutated viruses having altered dsRNA binding activity in cells and celllines which naturally exhibit an attenuated response to viral infectionsas compared to wild-type cells. In a particular preferred embodiment,the present invention relates to methods of growing the viruses of theinstant invention in Vero cells.

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method, kit, reagent, orcomposition of the invention, and vice versa. Furthermore, compositionsof the invention can be used to achieve methods of the invention.

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention.The principal features of this invention can be employed in variousembodiments without departing from the scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine experimentation, numerous equivalents to the specificprocedures described herein. Such equivalents are considered to bewithin the scope of this invention and are covered by the claims.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.” Throughout this application, the term “about” is used toindicate that a value includes the inherent variation of error for thedevice, the method being employed to determine the value, or thevariation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, MB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

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What is claimed is:
 1. An isolated crystal of protein complex comprisingresidues 85 to 215 of non-structural protein 1 (NS1) of influenza Avirus as set forth in SEQ ID NO: 1 and cellular polyadenylation andspecificity factor 30 (CPSF30) F2F3 fragment as set forth in SEQ ID NO:2, wherein the crystal has space group P4₁, and unit cell parametersa=b=50.96 Å, c=205.39 Å and α=β=γ=90°.
 2. An isolated protein complexcomprising non-structural protein 1 (NS1) of influenza A viruscomprising amino acids 85 to 215 of SEQ ID NO: 1 in complex withcellular polyadenylation and specificity factor 30 (CPSF30) F2F3fragment consisting of amino acids as set forth in SEQ ID NO:
 2. 3. Theisolated protein complex of claim 2, wherein the complex comprises atetramer interface.
 4. The isolated protein complex of claim 3, whereinthe tetramer interface comprises atoms of residues F103, L105, M106 ofinfluenza strain A/Udorn/72.
 5. The isolated protein complex of claim 2,wherein the complex comprises a CPSF30-binding epitope comprising M106,K110, I117, I119, Q121, D125, L144, V180, G183, G184, W187 of influenzastrain A/Udorn/72.